In the last two decades, there has been substantial interest in identifying antimicrobial peptides from a variety of donor organisms and exploiting their use for plant protection against both fungal and bacterial pathogens using a transgenic strategy. Among natural lytic peptides, cecropins, melittin and their hybrid chimeras have been tested extensively for their antimicrobial activity.
Cecropins were first found in the hemolyph of pupae of the Hyalophora cecropia silkmoth (Steiner et al. 1981) but subsequently identified from numerous insect species and even from the animal kingdom (Boman and Hultmark 1987; Lee et al. 1989). Structural analyses indicate that cecropins are usually composed of 35-39 amino acid residues with two different domains connected by a flexible non-helical hinge region. The N-terminal domain (head) contains a high proportion of basic amino acids and folds into a perfect amphipathic α-helix, while the C-terminal domain (tail) is rich in hydrophobic residues and forms a more hydrophobic helix (Van Hofsten et al. 1985). The charged N-terminal amphipathic α-helix can easily span a negatively-charged bacterial lipid membrane and exhibit voltage-dependent ion-permeable pore-forming properties (Christensen et al. 1988). Cecropins are potent lytic peptides capable of lysing in vitro a wide variety of Gram-positive and Gram-negative bacteria and play an important role in the humoral immune system in insects without known adverse activity against eukaryotic cells.
Elittin is a 26-residue peptide that constitutes a major toxic component of the venom of European honey bee Apis mellifera (Habermann 1972). Melittin also has a helix-hinge-helix structure similar to that of cecropins but with opposite polarity, i.e. a hydrophobic N terminus and an amphipathic C terminus. The powerful hemolytic and allergenic activity of melittin has precluded any practical use of this peptide as a whole for antimicrobial purposes, nonetheless, its potent antibacterial activity attracted great attention.
Extensive structure-function studies revealed that the amphiphilic helical N-terminal segment (residues 1-14) of melittin possesses channel-forming capability and thus is mostly responsible for antibacterial activity, whereas the hinge region plays a crucial role in modulating hemolytic activity. The C-terminal segment (residues 20 to 26) of melittin had no effect on lytic activity (Sitaram and Nagaraj 1999).
Hybrid peptides composed of various segments of cecropins and melittin have been synthesized and tested in vitro for biological activity against pathogenic bacteria. Based on structural analyses and the concept of α-helix-membrane interactions, Boman et al. (1989) first proposed the use of such hybrid peptides and examined several chemically synthesized hybrid peptides composed of the α-helix regions from different peptides. They found that chimeric peptides containing the amphiphilic 1-13 or 1-8 N-terminal segment of cecropin A and 1-13 or 1-18 region of melittin showed broad-spectrum antimicrobial activity that was up to 100-fold higher than the activity of natural cecropin A. Noticeably, these hybrid peptides, unlike melittin, had low hemolytic activity and did not lyse sheep red blood cells, even at 50-200 times higher concentrations (Boman et al. 1989 and Wade et al. 1990).
Another research group (Piers et al. 1994) subsequently modified the C terminus of a cecropin-melittin hybrid CEME (cecropin 1-8 plus melittin 1-18) that was previously created by Wade et al. (1990) to produce a peptide called CEMA (cecropin 1-8 plus melittin 1-16 plus KLTK). The C-terminal modification with charged residues KLTK in CEMA was suggested to have improved interactions between lytic peptide and lipid membrane or lipid membrane affinity for better outer membrane-permeabilizing capability (Piers et al. 1994). However, various studies by the same group showed that, in spite of the higher in vitro lipid membrane affinity of CEMA, both CEME and CEMA had an essentially identical level of antibacterial activity (Peiers et al. 1994; Gough et al. 1996; Scott et al. 1999) and that CEME also had a high-binding affinity to bacterial lipid membranes and an outstanding outer membrane-permeabilizing capability (Piers and Hancock 1994).
Active natural and synthetic lytic peptides including cecropins and melittin have an amidated C terminus (Wade et al. 1990; Andersons et al. 1991). The addition of charged residues to the C terminus of CEMA was also suggested to provide a charged environment in recombinant lytic peptides for efficacious interactions with lipid membranes (Piers et al. 1994). However, in a previous study, the same authors demonstrated that recombinant CEME lacking charged C-terminal residues produced by a bacterial expression system had properties (including antibacterial activity) identical to those of chemically synthesized CEME with amidated C terminus (Piers et al. 1993).
Details regarding the design and use of CEMA and related peptides can also be found in several recent US patents by Hancock et al. (U.S. Pat. Nos. 5,593,866; 5,707,855; 6,288,212 and 6,818,407).
Over the years, a variety of lytic peptides have been tested to confer resistance to phytopathogens in transgenic plants. However, the use of genes encoding natural peptides such as cecropins remained to be relatively ineffective and failed to confer any detectable resistance (Hightower et al. 1994; Florack et al. 1995), while genes coding for modified lytic peptides or analogues provided limited enhanced resistance (Arce et al. 1999). Recently, Osusky et al. (2000) reported the obtainment of broad-spectrum resistance in transgenic potato plants by using a variant of CEMA gene, MsrAl. This peptide was modified with a 6-residue (MALEHM) (residues 1 to 6 of SEQ ID NO:1) extension at the N terminus of CEMA. The addition of this hexapeptide was postulated to reduce toxicity of peptide products for the host plant. However, constitutive expression of the MsrAl gene in one of several tested potato genotypes resulted in the non-pathogen-induced “lesion-mimic” morphological changes (Osusky et al. 2000).